The sulfur analogue of adenosine 5'-monophosphate, adenosine 5'-O-thiomonophosphate (AMPS), contains a prochiral phosphorus center. Differentiation of the two diastereotopic oxygens would allow elucidation of the stereochemical course of biological adenylyl transfer reactions in which the oxygen from one of the substrates is incorporated into the product AMPS. We will develop 17O and 31P NMR methods to distinguish between the pro-R and pro-S oxygens. By converting the AMPS to the isomer A of adenosine 5'-O-(1-thiotriphosphate) (ATP-alpha-S), which is known to have "S" configuration at P-alpha, the pro-R oxygen is located at the bridge position whereas the pro-S oxygen is located at the non-bridge position. The 17O and 31P NMR spectra of the 17O-enriched compounds will be used to distinguish between the bridge and non-bridge oxygens. The 17O NMR method is based on the chemical shift difference whereas the 31P NMR method is based on the decrease in peak intensity caused by directly bound 17O isotope. Seven enzymes of different classes will be investigated: snake venom phosphodiesterase, acetyl CoA synthetase, argininosuccinate synthetase, asparagine synthetase, guanosine 5'-monophosphate synthetase, phenylalanyl-tRNA synthetase, and tyrosyl-tRNA synthetase.